Fungal infections are an emerging medical threat, particularly for immunocompromised individuals, yet little is understood about the molecular basis of immune evasion by fungal pathogens. The long-term goal of this research is to understand how intracellular fungal pathogens subvert host defenses. This aim interfaces with the fundamental goal of this program project application, which is to understand how diverse intracellular pathogens such as Francisella tularensis, Listeria monocytogenes, Legionella pneumophila, and Mycobacterium tuberculosis manipulate macrophage responses. Our model system is Histoplasma capsulatum, an intracellular fungal pathogen that, like Mycobacterium tuberculosis, survives and replicates in the phagosome of macrophages. Very little is known about how H. capsulatum colonizes a niche that is normally hostile to microbes. During infection, Histoplasma undergoes a morphologic switch from the infectious to the parasitic form, and ultimately establishes a chronic infection even in a healthy host. Our overarching hypothesis is that there are molecular determinants of pathogenesis that are produced by Histoplasma at different stages of infection, and that the development and outcome of disease are influenced by the host response to these stage-specific determinants. Our specific aims are to (1) use molecular genetics approaches to identify and characterize specific virulence pathways utilized by Histoplasma to colonize macrophages; (2) use whole-genome expression profiling to characterize the macrophage response to the infectious and parasitic forms of the organism; and (3) use the mouse model of histoplasmosis to determine the role of specific Histoplasma virulence determinants (characterized in Aim 1) and host signaling pathways (characterized in Aim 2). A comparative analysis of the resulting data with the other program project organisms will identify regulatory circuits in host cells that are manipulated by H. capsulatum and other key intracellular pathogens relevant to biodefense and emerging infections. Relevance to public health: H. capsulatum is a primary pathogen that infects approximately 500,000 individuals per year in the U.S. and is a significant source of morbidity and mortality in immunocompromised patients. Since very little is understood about how it causes disease, the identification of fungal and host factors that influence pathogenesis will significantly advance the field and provide fodder for the development of new therapeutics.